Transcription factor for improving plant seed fatty acid content, coding gene and application thereof
A fatty acid and encoding technology, applied in the direction of plant genetic improvement, application, plant peptides, etc.
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Embodiment 1
[0073] Embodiment 1, the acquisition of the coding gene GhDof1 of cotton transcription factor GhDof1
[0074] 1. Total RNA extraction: Upland cotton (Gossypium hirsutum L.) variety Coker201 (Wu Xiuming, Liu Chuanliang, Zhang Chaojun, Li Fuguang. Research progress in cotton somatic embryogenesis. Bulletin of Botany, 2008,25(4):469-475 ) as the experimental material, the immature embryo material 20 days after flowering was taken, quick-frozen in liquid nitrogen, and CTAB method was applied (references: Liu Yang, He Xinyao, Ma Hongbo, Wu Yongli, Yang Youming. The total RNA of each tissue of cotton was extracted by CTAB-PVP method Research. Journal of China Agricultural University, 2006, 11(1):53-56) to extract RNA.
[0075] 2. Use Promaga's RT-PCR reaction system to reverse transcribe the RNA extracted in step 1 to synthesize cDNA.
[0076] 3. RT-PCR amplification:
[0077] According to the EST information of cotton in the NCBI database, spliced into contig, the following pri...
Embodiment 2
[0085] Example 2, Functional Analysis of Transcriptional Activation of GhDof1 Transcription Factor
[0086] In this example, pGBT9 (Meng Hongyan, Du Xiongming, Zhang Chunyi, etc. Cloning of cotton transcription factor gene GhMS3 and identification of its promoter function. Chinese Agricultural Sciences, 2010, 43 (17)) was used as the original vector to construct the following three expression vectors ( figure 2 Middle A): (1) the recombinant expression vector pGBT-GhDof1 containing the full-length CDS of the GDof1 gene (sequence 1, referred to as GDof1-CDS); (2) containing the N-terminal of the GDof1 gene (the 1-501st nuclear (GDof1-N for short) recombinant expression vector pGBT-GhDof1-N; (3) recombinant expression vector pGBT containing the C-terminal of the GhDof1 gene (nucleotides 481-948 of sequence 1, GhDof1-C for short) -GhDof1-C. In order to analyze the transcription activation function of GhDof1 transcription factor.
[0087] 1. Construction of recombinant express...
Embodiment 3
[0102] Embodiment 3, expression analysis of GhDof1 gene
[0103] RNA was extracted from the ovary, anther, calyx, petal, ovule, root, stem and leaf tissue of upland cotton (Gossypium hirsutum L.) variety Coker201, and cDNA was synthesized by reverse transcription with Promaga's RT-PCR reaction system, and designed for the GhDof1 gene Primers 5'-TGATGTGAGTGAACCGAAGT-3' (positions 507-526 of Sequence 1) and 5'-TTTACGGGAGATAGAGGC-3' (positions 2-18 are the reverse complement of positions 932-948 of Sequence 1) to Ghactin As an internal reference gene, primers 5'-ATCCTCCGTCTTGACCTTG-3' and 5'-TGTCCGTCAGGCAACTCAT-3' were designed, and the expression of GhDof1 gene in different tissue parts was analyzed by semi-quantitative RT-PCR.
[0104] The result is as Figure 4 As shown, the GhDof1 gene exhibits certain tissue specificity, which is specifically manifested in: the gene is expressed in ovary, anther, calyx and other reproductive organ tissues, while the expression level in peta...
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